Alkaline, protease VapK suitable for laundry detergent, VapK gene, recombinant expression vector, and transformed microorganisms

ABSTRACT

A novel alkaline protease VapK suitable for a laundry detergent is disclosed. The gene vapk coding for the protease VapK, the recombinant plasmids containing said gene, and the transformed  V. metshnikovii  KS1 (pSBCm) with said recombinant plasmid are also disclosed. In addition, a process for producing the protease VapK is disclosed.

This application is the national phase under 35 U.S.C. § 371 of PCTInternational Application No. PCT/KR00/00022 which has an Internationalfiling date of Jan. 14, 2000, which designated the United States ofAmerica.

FIELD OF THE INVENTION

The present invention relates to an alkaline protease VapK suitable fora laundry detergent, produced by Vibrio metschnikovii KS1, and to thegene vapk coding for said protease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the restriction map of the recombinant plasmid pSB1 of thepresent invention.

FIG. 2 shows the restriction map of the recombinant plasmid pSBCm of thepresent invention.

FIG. 3 shows the results of the Southern blotting of the vapk gene.

-   -   A: Agarose gel electrophoresis of plasmid pSB1 and V.        metschnikovii KS1 chromosomal DNA digested by Hind III (lane        1: V. metschnikovii KS1 chromosomal DNA/Hind III; lane 2: V.        metshnikovii KS1 chromosomal DNA; lane 3: pSB1 (super-coiled        form); lane 4: pSB1/Hind III; lane 5: pT7T3/Hind III; lane 6:        Pseudomonas sp. BK7; M: λDNA/Hind III)    -   B: Southern hybridization of DIG-labeled insert (0.7 kb) of pSB1        and V metschnikovii KS1 chromosomal DNA (lane 1: V. metshnikovii        KS1 chromosomal DNA/Hind III; lane 2: V. metshnikovii KS1        chromosomal DNA; lane 3: pSB1 (super-coiled form); lane 4:        pSB1/Hind III; lane 5: pT7T3/Hind III; lane 6: Pseudomonas sp.        BK7; M: λ DNA/Hind III).

FIG. 4 illustrates the subcloning for identifying the minimum size ofthe vapk gene.

FIG. 5 shows the nucleotide sequence (SEQ ID NO:2) of the vapk gene andthe deduced amino acid sequence (SEQ ID NO:1), wherein the symbol “=”indicates the mature form of N-terminal region identified by analysis ofthe N-terminal sequence and the symbol “*” indicates the active site.

FIG. 6 shows the comparison of the amino acid sequences of VapK (Vibrioalkaline protease K)(SEQ ID NO:3), VapT (Vibrio alkaline protease T)(SEQID NO:4), BAE (Bacillus sp. elastase TaB)(SEQ ID NO:5), and CAR(Bacillus licheniformis subtilisin Carlsberg)(SEQ ID NO:6), in which thesymbol “*” indicates the active site.

FIG. 7 shows the results of SDS-PAGE of the recombinant VapK expressedin E. coli [lane 1: V. metschnikovii culture broth; lanes 2 and 4: E.coli (pSB1) culture broth; lane 3: crude extract of E. coli (pSB1)culture cells; lane M: protein size markers indicating molecularweights].

FIG. 8 shows the optimum pH and temperature of the recombinant VapK.

BACKGROUND OF THE INVENTION

Enzymes used as laundry detergents have been continually improved sincetheir commercialization in the 1970s. To date, protease, lipase,amylase, and cellulase have been developed for laundry detergents. Amongthese, protease has been the most widely used as an additive of laundrydetergent. Widely used alkaline protease for laundry detergents exhibittheir optimal activity at pHs 8 to 12 and at temperature of 40° C. to60° C. Most proteases used as detergent enzymes are produced by theBacillus species. They are classified as a serine protease and arewidely known as subtilisin.

Subtilisin exhibits stability in high temperature but is not stableunder certain conditions. Specifically, its activity is not maintainedfor a prolonged period upon exposure to urea, a protein denaturizingagent. Subtilisin also quickly and irreversibly loses its proteolyticactivity at pH 4 or below. Moreover, the activity of subtilisin is weakat low temperature (15° C. to 25° C.). As such, it cannot be usedeffectively in Southeast Asia and Central and South America where peopleusually wash with low temperature water.

U.S. Pat. Nos. 5,741,694 and 5,482,849 describe subtilisin withbiochemical properties altered through a site-directed mutagenesis. U.S.Pat. No. 5,401,657 describes bacteria which produce an alkali-resistantenzyme useful as a laundry detergent.

We isolated a vapk gene encoding a protease enzyme VapK, which can beadvantageously used in laundry detergent in that it remains active athigh temperature and at high alkaline pH, and is resistant to manysurfactants and protein denaturants widely used in laundry detergents.The gene vapk has been cloned, sequenced and brought to expression inuseful host cell. The gene vapk consists of 1,266 bp coding for 422amino acids, and the molecular weight of the protease VapK is 27 kda.This enzyme is very resistant to various surfactants such as AOS or LASand exhibits optimum activity at pH 10.5 and at the temperature of 50°C. Moreover, the protease VapK is more active at low temperatures thanare commercially availiable enzymes.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel protease VapK having thefollowing amino acid sequence (SEQ ID NO. 1):

Met Phe Lys Lys Asn Val Asn Arg Thr Val Leu Ala Gly Leu Leu Leu1               5                   10                  15 Pro Thr SerIle Ser Leu Ala Ile Ala Ser Gln Leu Lys Asp Gln Glu            20                  25                  30 Val Pro Ser PheThr Pro Ser Val Ala Val Glu Asn His Gln Thr Glu        35                  40                  45 Gln Arg Tyr Phe ValThr Tyr Val Pro Gly Ala Thr Ser Gly Pro Met    50                  55                  60 Arg Met Ser Gln Asn GlyLeu Thr Glu Thr Asp Phe Ser Leu Gln Lys65                  70                  75                  80 Ala AlaAsp Ile Leu Ser Thr Gln Gln Val Thr Val Ile Asn His Leu                85                  90                  95 Glu Ser LeuHis Thr Ser Val Val Arg Val Thr Pro Thr Gln Ala Lys            100                 105                 110 Gln Leu Leu AspAsn Ala Asp Val Ala Met Ile Glu Val Asp Pro Ile        115                 120                 125 Arg Tyr Leu Phe AspAla Glu lle Glu Pro Tyr Ala Gln Gln Thr Pro    130                 135                 140 Tyr Gly Ile Arg Met ValGln Ala Asp Gln Leu Ser Asp Val Tyr Ala145                 150                 155                 160 Ala AsnArg Lys Val Cys Val Ile Asp Ser Gly Tyr Leu Arg Asn His                165                 170                 175 Val Asp LeuPro Ser Ala Gly Val Thr Gly Ser Thr Phe Ser Gly His            180                 185                 190 Gly Ser Trp PheThr Asp Gly Asn Gly His Gly Thr His Val Ala Gly        195                 200                 205 Thr Ile Val Ala LeuAsp Asn Asn Val Gly Val Val Gly Val Leu Pro    210                 215                 220 Ser Gly Leu Val Gly LeuHis Asn Val Lys Ile Phe Asn Asp Ser Gly225                 230                 235                 240 Val TrpThr Arg Ala Ser Asp Leu Ile Gln Ala Ile Gln Ser Cys Gln                245                 250                 255 Ser Ala GlySer His Val Val Asn Met Ser Leu Gly Gly Ser Gln Gly            260                 265                 270 Ser Val Thr GluGln Lys Pro Met Arg Asn Phe Tyr Gln Gln Gly Met        275                 230                 285 Leu Leu Val Ala AlaAla Gly Asn Ser Gly Asn Ser Gly Phe Ser Tyr    290                 295                 300 Pro Ala Ser Tyr Asp AlaVal Val Ser Val Ala Ala Val Asn Ser Ser305                 310                 315                 320 Gly AsnVal Ala Asn Phe Ser Gln Phe Asn Ser Gln Val Glu Leu Ser                325                 330                 335 Ala Pro GlyVal Gly Val Leu Ser Thr Gly Asn Asn Gly Gly Tyr Leu            340                 345                 350 Ser Tyr Ser GlyThr Ser Met Ala Ser Pro His Val Ala Gly Val Ala        355                 360                 365 Ala Leu Val Trp SerHis Phe Pro Gln Cys Arg Pro Glu Arg Ile Arg    370                 375                 380 Gln Ser Leu Ser Gln ThrAla Leu Asp Arg Gly Ala Ala Gly Arg Asp385                 390                 395                 400 Asn PheTyr Gly Trp Gly Ile Val Gln Ala Arg Arg Ala Tyr Asn Trp                405                 410                 415 Leu Ser ArgAsn Gly Cys             420

The present invention also provides a vapk gene having the followingnucleotide sequence (SEQ ID NO. 2):

agcttctaat acgactcact atagggaaag ctttgctttc ggttttttct gccgcttgac 60agatatttat gcgattcata atggataaat aatcattata aatcgccatg atgtaaatca 120agtagactaa aaaacgtaca gtttttttta cttaatagtc tatcaatatc attaatttaa 180ccaataggta acaattcagt aaataaaagc aaacacattc acccagctaa taatagcttt 240attaataaac tataaaactg gcgaatggct gccgttgtaa tgatccttga tgagtggtat 300ttgccactaa cattttacaa ggataaaaaa atgttgaaga aaaatgtcaa ccgtacagta 360ctggctgggt tattgttgcc aacttcaatc tcactggcaa tagcatctca gcttaaggat 420caagaagtac cgagttttac cccctctgtt gctgttgaaa atcatcaaac agaacaacgc 480tattttgtta cctacgtgcc tggggcaacc agcggaccaa tgcggatgag tcaaaacggc 540ttaacagaaa cagatttctc tctgcaaaaa gccgccgata tattaagtac tcagcaagta 600acggtcatca atcacctcga gtcattacat acttcagtgg ttagagtgac gccaactcaa 660gccaagcaac tgctcgataa tgctgatgtg gcgatgatcg aagtcgaccc aatacgctat 720ttattcgatg ctgagattga gccttacgca caacagaccc catacggaat ccgtatggta 780caagccgatc aactctctga cgtttatgcg gctaatcgta aactttgcgt catcgactcg 840ggatatcttc gcaaccatgt tgatctaccg agcgctggag tcacaggcag cactttctct 900ggccatggtt catggttcac tgatggcaat ggtcatggaa ctcacgttgc agggacaatt 960gtcgcactgg ataataatgt cggagttgtt ggggttctac cgtctggctt agtcggccta 1020cacaacgtaa aaatctttaa cgattccggt gtctggactc gcgcttcgga tttgattcaa 1080gctatccaat cttgtcaaag tgcaggcagt catgtggtaa atatgagttt aggtggtagc 1140caaggcagtg taaccgaaca aaagccaatg cgtaactttt accaacaagg gatgctctta 1200gttgcagcag caggtaactc aggaaacagc ggcttctcat acccggcgtc ttatgatgca 1260gtggtctcag ttgcggcggt taactcaagt ggtaatgtgg ctaacttctc acagttcaat 1320tcacaagttg aactctcggc accaggagtt ggggtactat caaccggtaa taatggcggc 1380tacttaagct atagcggaac ctcaatggct tcacctcacg ttgcaggtgt cgcagcgctg 1440gtttggagtc actttccaca atgtcgacca gagcgaatcc gtcagtcact cagtcaaacg 1500gctctcgatc gtggtgccgc aggtagagat aatttttacg gttgggggat agttcaagcg 1560agacgtgcct ataactggtt atctcgcaat ggctgttaat ttctaatatt gagaatatga 1620acagggtact gagtaccctg tttgttattt tccagagaca aatctaaccg tctaaattaa 1680tttgtttaat caattcttct tgtacacggt ctgcctcaga taagggaatt tgacaggttc 1740cagcggcatg atgaccgccg ccaccatatt tgagcattaa cgcaccaaca ttggtttgga 1800actacgatct ttc 1813

In addition, the present invention provides a recombinant plasmid pSB1containing the vapk gene and expressing it.

Further, the present invention provides a recombinant plasmid pSBCmcontaining the vapk gene and expressing it in a high yield.

Furthermore, the present invention provides a Vibrio metschnikovii KS1(pSBCm) formed by transformation of V. metschnikovii KS1 with therecombinant plasmid pSBCm.

The present invention also provides a process for producing the proteaseVapK comprising the steps of culturing the V. metschnikovii KS1 (pSBCm)under conditions which allow the expression of protease VapK, andpurifying it from the culture broth.

The characteristics of the recombinant plasmids pSB1 and pSBCm accordingto the present invention and the processes for producing said plasmidsare described below.

Recombinant Plasmid pSB1 (FIG. 1)

For cloning the vapk gene, chromosomal DNA of V. metschnikovii KS1 waspartially digested and inserted into a vector pT7T3 19U. The resultingrecombinant vectors were introduced into a host cell. The transformedmicroorganisms were cultured on a solid agar medium containing skim milkand the transformant, showing a clear halo, was isolated. The plasmidwas isolated and identified from the transformant. The identifiedplasmid was revealed that it contains a 3.0 kb DNA insert and two HindIII sites. The E. coli cells having the isolated plasmid DNA werecultured. The alkaline protease in the culture solution was analyzed anddetermined to have a molecular weight of 27 kDa and to exhibit optimumactivity at pH 10.5.

Recombinant Plasmid pSBCm (FIG. 2)

The recombinant plasmid was transformed into V. metschnikovii KS1 toincrease the productivity of the alkaline protease. Therefore, selectionfactors, such as antibiotic-resistance or colony-discoloration, wereused to isolate the clone. The 3.0 kb vapk gene contained in plasmidpSB1 was subcloned into vector pKF3 which includes the cat-5 gene forchloramphenicol resistance (Cm). V. metschnikovii KS1 is sensitive tothe antibiotic chloramphenicol. Therefore, it is easy to select theclone formed by transforming V. metschnikovii KS1 with the recombinantvector. The ampicilin-resisting factor is not suitable for the selectionof the clone because V. metschnikovii KS1 shows resistance to theantibiotic ampicilin. The plasmid pSB1 was digested with Hind III andthe resulting fragment was subcloned into the vector pKF3 digested withthe same restriction enzyme Hind III. The recombinant vector obtainedtherefrom was introduced into E. coli. A strain exhibiting resistance tochloramphenicol and showing halo arround the colony on the skim-milkplate was isolated. The isolated strain contains the recombinant plasmidpSBCm. This pSBCm vector was used in transforming V. metschnikovii KS1.

In addition to V. metschnikovii KS1, E. coli HB101, E. coli JM101, E.coli Top10F′, V. metschnikovii RH530 N4-8, etc. can be used as a host.

To produce the desired alkaline protease, the transformed V.metschnikovii KS 1 was cultured in an LSC medium consisting of 1%Bacto-tryptone, 0.5% yeast extract, 1% sodium chloride, and 100 mMsodium carbonate buffer solution, pH 10.5.

The activity of the alkaline protease was determined by the method ofYanagida et al with minor modifications. The reaction mixture wasprepared by mixing 100 mM sodium carbonate solution with the supernatantformed after centrifuging the culture broth at 6,000 rpm for 15 minutes.0.5 ml of the fermentation broth was mixed with 2.5 ml of 1% prewarmedcasein solution in 100 mM sodium carbonate buffer (pH 10.5). Theresulting mixture was incubated at 37° C. for 10 minutes. The reactionwas terminated by adding a solution consisting 0.22 M trichloroaceticacid, 0.22 M sodium acetate and 0.22 M acetic acid. The reaction mixturewas placed on ice for 10 minutes followed by centrifugation. 1 ml of thesupernatant was mixed with 9 ml of distilled water. The optical density(O.D.) was measured at 280 nm. The result is indicated as unit. One unitof the enzyme is defined as the amount of protein which produces anincrease of 0.1 absorbance unit under the assay conditions.

The gene sequencing was conducted by a dideoxy chain termination method(Sanger et al., 1997, Proc. Natl. Acad. Sci. USA. 74: 5463-5467).

The invention will now be described with reference to the followingillustrative Examples.

EXAMPLES Example 1 Construction of Recombinant Plasmid pSB1

The recombinant plasmid pSB1 was constructed by a shot-gun cloningmethod. After growth of V. metschnikovii KS1, an alkalineoverproducing-mutant, its chromosomal DNA was separated by the Mamurmethod (Mamur, et al., J. Mol. Biol. 3:208-218, 1961). The chromosomalDNA was partially digested with Hind III, and the fragment wasprecipitated with ethanol and recovered. The plasmid pT7T3 19U wasprepared by the Birnboim and Doly method (Nucleic acids Res.7:1513-1523, 1979), and digested with Hind III. Both DNAs were ligated.The resulting recombinat plasmid was introduced into E. coli HB101. Thetransformant was plated on an LB-Ap-skim milk medium containing 1%bacto-tryptone, 0.5% bacto-yeast extract, 1% NaCl, 40 μg/ml ofampicilin, and 1% skim milk. Several colonies, each forming a clear haloaround the colony, were selected as the clones possessing the alkalineprotease gene. One of them was confirmed as harboring the vapk geneencoding the alkaline protease VapK. The recombinant plasmid with thevapk gene was designated as pSB1. The nucleotide sequence of the vapkgene of pSB1 and the amino acid sequence of the expressed alkalineprotease VapK were determined. In addition, the properties of thealkaline protease VapK were studied.

Example 2 Identification of the Recombinant Plasmid pSB1

FIG. 1 shows the restriction map of the recombinant plasmid pSB1. Theplasmid pSB1 is 5.9 kb in length and contains 3.0 kb vapk gene. ASouthern blotting with the chromosomal DNA of V. metschnikovii KS1 wasperformed with the 3.0 kb vapk gene probe. As a result, it was concludedthat the cloned gene was derived from V. metschnicovii KS1. In addition,the recombinant vapk gene was digested with various restriction enzymesincluding Bal 31 exonuclease. As a result, it was found that at least2.1 kb of the vapk gene is required for encoding the alkaline proteaseVapK (FIG. 4).

Example 3 Illustration of the Nucleotide Sequence of the vapk Gene

The 3.0 kb vapk gene was digested with various restriction enzymes. Eachof the resulting fragments was subcloned into plasmid pUC19. Theoverlapping sites were drawn. The nucleotide sequence was determinedbased on such overlapping sites (SEQ ID Nos 1 and 2, and FIG. 5). Asingle open reading frame (ORF) of 1,266 bp exists between base 331 andbase 1,596. The real molecular weight of the protease VapK is 27 kDa asmentioned above. However, the MW of the enzyme deduced from the ORF is45 kDa. The difference results from the processing of the precursorregion during the extracellular secretion of the protease VapK.

The precursor region is the signal sequence inducing the extracellularsecretion of the expressed protein and consists of 25 amino acids. Thesite between Ala 25 and Ser 26 is expected to be processed. The aminoacid positions 3 and 4 which are highly basic amino acids are lysine.The amino acid positions 5 through 25 include strongly hydrophobic aminoacids. The −35 region (5′TTGACA3′), −10 region (5′TATAAAT3′) andShine-Dalgarno (SD) sequence (5′CAAGGA3′) located 8 bp upstream of theinitiation codon (ATG) are similar to those previously reported (FIG.5). The dyad symmetrical sequence, located 18 bp downstream of the stopcodon TAA, forms a stem-loop structure and functions as a rho (ρ)independent terminator. The amino acid sequence of the protease VapKdeduced from the nucleotide sequence of the vapk gene was compared to avariety of known proteases (FIGS. 6 and 7).

As shown in FIGS. 6 and 7, the protease VapK has 8%, 29%, and 29%nucleotide sequence homology with VapT, TaB, and Bacillus subtilisinCarlsberg, respectively. In addition, it is observed that His-202,Asp-169 and Ser-358 are well conserved as the active sites of allproteases (FIGS. 6 and 7).

Example 4 Characteristics of the Protease VapK Expressed from theRecombinant Plasmid pSB1

E. coil HB101 harboring the plasmid pSB1 was cultured in an LB medium.The culture broth was recovered and subjected to electrophoresis. As aresult (FIG. 8), the expressed protease VapK was detected at the 27 kDaband. In addition, the proteolytic activity of the enzyme was measured.The results are shown in Table 1 below.

TABLE 1 The proteolytic activity of expressed Vap K Strain Activity(unit/ml) Vibrio metschnikovii KS1 956.2 E. coli HB 101 30.3

It can be seen from the above results that the vapk gene contained inthe recombinant plasmid pSB1 was expressed in E. coli HB 101 and theexpressed protease VapK was secreted extracellularly. The protease VapKexhibited optimum activity at pH 10.5 and at 50° C. (FIG. 9). Thisenzyme showed stability in the entire pH range. However, the stabilityof the enzyme was influenced slightly by temperature. The protease VapKexerted high resistance to surfactants such as LAS (linear alkylbenzensulfonate), SDS (sodium dodecyl sulfonate), and AOS (Sodium-α-olefinsulfonate), making it suitable as a laundry detergent. The additionalcharacteristics of the expressed recombinant VapK are shown in Table 2below.

TABLE 2 Effects of Inhibitors on the activity of the protease VapKRelative Activity (%) Concentration of Temperature Inhibitor Inhibitor(mM) 4° C. 20° C. Metal chelating agent, 100 86 100 EDTA Serine proteaseinhibitor, 1 6 13 PMSF Reducing agent, L-cysteine 1 99 100 Calciumchelating agent, 1 100 100 EGTA EDTA: ethylenediamine tetraacetic acid,PMSF: phenylmethanesulfonyl fluoride, EGTA:ethyleneglycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid. *Theenzyme solution was previously reacted with each reagent at the giventemperature for 20 minutes.

The results indicated that the enzymatic activity of the protease VapKwas inhibited only by PMSF, the serine protease inhibitor. Thus, theprotease VapK is a serine protease. In addition, the enzymatic activityof the protease VapK at the temperature 4° C. was nearly identical withthat at 20° C. That is, the enzyme VapK showed high activity at lowtemperature. Accordingly, the protease VapK is an advance over proteasesused to date which are active only at high temperature.

In addition, the resistance of the protease VapK to various surfactantswas tested. The enzyme sample was appropriately diluted in distilledwater. The diluted solution was mixed with each surfactant in the sameratio. The mixture was reacted at 25° C. for 25 minutes. A substratesolution of 5 mM N-succinyl-ala-ala-pro-phe-p-nitroanilide in 1 Mtris-HCl, pH 9.0 was added to the reaction mixture in the ratio of 1 to10. The reaction mixture was reacted at 25° C. for 30 minutes. Theoptical density of the reaction mixture was measured at 410 nm todetermine the enzymatic activity of the protease VapK. The results areshown in Table 3 below.

TABLE 3 Concentration Relative Residual Surfactant (ppm) Activity^((a))(%) SDS^((b)) 1000 >100 1500 <70 AOS^((c)) 10000 >100 15000 >70Polyoxyethylene Alkylether^((d)) 30000 >100 (EO = 15 mol) ^((a))Valuerelative to the activity of the enzyme measured in the absence ofsurfactant ^((b)) SDS: sodium dodecyl sulfate (anionic surfactant)^((c)) AOS: Sodium-α-olefin sulfonate (anionic surfactant) ^((d))Nonionic surfactant.

The above results demonstrate that the protease VapK is very stable toanionic surfactants such as SDS and AOS and to nonionic surfactants suchas polyoxyethylene alkylether. Under general washing conditions, theamount of surfactant is no more than 1,000 ppm. Therefore, it isbelieved that the protease VapK remains stable to surfactants duringwashing.

Example 5 Transformation and Expression of the Recombinant Plasmid pSBCmto V. metschnikovii KS1

The V. metschnikovii KS1 was transformed with the recombinant plasmidpSBCm (FIG. 2). Vibrio strains secrete large amount of DNaseextracellularly or within the periplasmic space. Furthermore,when thestrains are shocked by high temperature, they easily die, even thoughthe DNase is inhibited. Thus, the transformation could not be easilyachieved by a conventional heat-shock method. Instead, thetransformation was performed by electroporation under the followingconditions:

(1) Effect of the Capacitance

The transformation efficiency was tested by varifying the capacitance inthe range of 1 μF to 50 μF. The survival rate was 96% at 1 μF and 9% at50 μF. The survival rate decreased as the capacitance increased. Thetransformation efficiency was highest at 10 μF.

(2) Effect of the Electric Field Strength

The transformation efficiency was tested by varifying the electric fieldstrength in the range of 5 kV/cm to 10 kV/cm while fixing thecapacitance at 10 μF. As a result, the highest transformation efficiencywas obtained at 7.5 kV/cm.

(3) Effect of the DNA Concentration

The transformation efficiency was tested by varifying the DNAconcentration in the range of 10 ng to 5 μg while fixing 10 μF ofcapacitance and 10 kV/cm of electric field strength. As a result, thetransformation efficiency increased in proportion to the DNAconcentration. In addition, the enzyme activity of the transformed V.metschnikovii KS1 (pSBCm) was tested. As shown in Table 4 below, theactivity of the transformed V. metschnikovii KS1 (pSBCm) was twice ashigh as that of the host strain V. metschnikovii KS 1.

TABLE 4 Microorganism Activity (unit/ml) E. coli HB 101 — V.metschnikovii KS1   900 V. metschnikovii KS1 (pSBCm) 1500Deposition of Microorganisms and Recombinant Plasmids of the PresentInvention

The strain V. metschnikovii KS1 of the present invention was depositedwith the Korean Culture Center of Microorganisms, Seoul, Korea, on Dec.15, 1998, as Accession No. KCCM-10141.

Also, the stain E. coli Top10F′ containing the recombinant vector pSBCmof the present invention was deposited with the Korean Culture Center ofMicroorganisms on Dec. 15, 1998, as Accession No. KCCM-10142.

1. An isolated alkaline protease gene comprising the nucleotide sequenceof SEQ ID NO:2: agcttctaat acgactcact atagggaaag ctttgctttc   40ggttttttct gccgcttgac agatatttat gcgattcata    80 atggataaat aatcattataaatcgccatg atgtaaatca   120 agtagactaa aaaacgtaca gtttttttta cttaatagtc  160 tatcaatatc attaatttaa ccaataggta acaattcagt   200 aaataaaagcaaacacattc acccagctaa taatagcttt   240 attaataaac tataaaactg gcgaatggctgccgttgtaa   280 tgatccttga tgagtggtat ttgccactaa cattttacaa   320ggataaaaaa atgttgaaga aaaatgtcaa ccgtacagta   360 ctggctgggt tattgttgccaacttcaatc tcactggcaa   400 tagcatctca gcttaaggat caagaagtac cgagttttac  440 cccctctgtt gctgttgaaa atcatcaaac agaacaacgc   480 tattttgttacctacgtgcc tggggcaacc agcggaccaa   520 tgcggatgag tcaaaacggc ttaacagaaacagatttctc   560 tctgcaaaaa gccgccgata tattaagtac tcagcaagta   600acggtcatca atcacctcga gtcattacat acttcagtgg   640 ttagagtgac gccaactcaagccaagcaac tgctcgataa   680 tgctgatgtg gcgatgatcg aagtcgaccc aatacgctat  720 ttattcgatg ctgagattga gccttacgca caacagaccc   760 catacggaatccgtatggta caagccgatc aactctctga   800 cgtttatgcg gctaatcgta aactttgcgtcatcgactcg   840 ggatatcttc gcaaccatgt tgatctaccg agcgctggag   880tcacaggcag cactttctct ggccatggtt catggttcac   920 tgatggcaat ggtcatggaactcacgttgc agggacaatt   960 gtcgcactgg ataataatgt cggagttgtt ggggttctac1000 cgtctggctt agtcggccta cacaacgtaa aaatctttaa 1040 cgattccggtgtctggactc gcgcttcgga tttgattcaa 1080 gctatccaat cttgtcaaag tgcaggcagtcatgtggtaa 1120 atatgagttt aggtggtagc caaggcagtg taaccgaaca 1160aaagccaatg cgtaactttt accaacaagg gatgctctta 1200 gttgcagcag caggtaactcaggaaacagc ggcttctcat 1240 acccggcgtc ttatgatgca gtggtctcag ttgcggcggt1280 taactcaagt ggtaatgtgg ctaacttctc acagttcaat 1320 tcacaagttgaactctcggc accaggagtt ggggtactat 1360 caaccggtaa taatggcggc tacttaagctatagcggaac 1400 ctcaatggct tcacctcacg ttgcaggtgt cgcagcgctg 1440gtttggagtc actttccaca atgtcgacca gagcgaatcc 1480 gtcagtcact cagtcaaacggctctcgatc gtggtgccgc 1520 aggtagagat aatttttacg gttgggggat agttcaagcg1560 agacgtgcct ataactggtt atctcgcaat ggctgttaat 1600 ttctaatattgagaatatga acagggtact gagtaccctg 1640 tttgttattt tccagagaca aatctaaccgtctaaattaa 1680 tttgtttaat caattcttct tgtacacggt ctgcctcaga 1720taagggaatt tgacaggttc cagcggcatg atgaccgccg 1760 ccaccatatt tgagcattaacgcaccaaca ttggtttgga 1800 actacgatct ttc
 1813.


2. A recombinant plasmid pSB1 containing the gene of claim
 1. 3. Arecombinant plasmid pSBCm containing the gene of claim
 1. 4. A strainVibrio metschnikovii KS1 transformed with the recombinant plasmid pSBCmof claim
 3. 5. A process for producing a protease VapK suitable for alaundry detergent which comprises cultivation of the strain of claim 4under conditions permitting the expression of protease VapK andpurifying the protease VapK from the culture.